MB-qPCR detection kit and detection method for rapidly detecting enterocytozoon hepatopenaei

Abstract

The invention discloses an MB-qPCR detection kit and a detection method for rapidly detecting enterocytozoon hepatopenaei, and belongs to the technical field of agricultural and aquatic organism detection. The kit comprises Tris-HCl, dNTP (deoxyribonucleoside triphosphate) containing U, KCl, MgCl2, Tween-20, tetrahydrothiophene 1-oxide, tetramethylammonium chloride, betaine, DMSO (dimethyl sulfoxide), Triton X-100, an upstream primer with a sequence as shown in SEQ ID NO. 1, a downstream primer with a sequence as shown in SEQ ID NO. 2 and an MB fluorescent probe with a sequence as shown in SEQ ID NO. 3. The invention also provides a method for detecting enterocytozoon hepatopenaei by using the kit, the enterocytozoon hepatopenaei can be rapidly detected, and the kit also has the characteristics of high sensitivity, strong specificity and wide detection range.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural and aquatic biological detection, and in particular to an MB-qPCR detection kit and a detection method for rapid detection of shrimp hepatoenterozoa. Background Art

[0002] Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasitic pathogen discovered in the shrimp farming industry in recent years. It is easy to infect major economically farmed shrimps such as Penaeus vannamei, white shrimp and Penaeus monodon, and is one of the important pathogens affecting global shrimp farming production. Individuals infected with EHP have no obvious symptoms in the early stage, slow movement and bottom lying, lethargy, decreased appetite, decreased stress resistance, and loose shells in the middle stage; after 2-3 months of infection, the growth rate of diseased shrimp slows down or even stops, and its body size is only half the size of healthy shrimp. Some diseased shrimp have symptoms of white stool. Although shrimp infected with EHP will not die acutely, the shrimp will stagnate and the symptoms will worsen with the increase of infection time, which may cause chronic death of shrimp, seriously affecting the shrimp farming industry and food safety, thus leading to large economic losses.

[0003] At present, the detection technology of shrimp hepatoenteric cysts has made great progress, and the histological detection technology and molecular biological detection technology of shrimp hepatoenteric cysts have been established. For example, in Chinese invention patent 201710917654.4, a fluorescent kit for detecting shrimp hepatoenteric cysts is provided by a constant temperature amplification method. Although there is no need for precise experimental instruments and equipment, its specificity is low and it is easy to have problems such as false positives; in Chinese invention patent 201811501504.6, a set of primers is designed based on the conservative region of the SWP1 gene of shrimp hepatoenteric cysts, but agarose gel electrophoresis is required after PCR amplification, which is easy to cause aerosol contamination during the opening process, and the method has a high detection limit, is prone to false negatives, and is time-consuming and labor-intensive. Therefore, it is of great significance and application value to design an efficient and practical detection kit for shrimp hepatoenteric cysts. Summary of the invention

[0004] In view of the defects of the prior art, the present invention designs a MB-qPCR detection kit for rapid detection of shrimp hepatoenterozoa and a detection method using the kit, which has the characteristics of simple operation, high specificity, high sensitivity and no aerosol pollution.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: On the one hand, the present invention designs an MB-qPCR detection kit for rapid detection of shrimp hepatoenteroides based on MB-qPCR technology. MB-qPCR is simple to operate, sensitive, and can perform real-time amplification detection on samples. Compared with ordinary linear oligonucleotide probes, the molecular beacon with a stem-loop structure can detect mismatches, deletions, or insertion mutations of single bases in the target sequence.

[0006] The kit includes MB-qPCR Mix, Enzyme Mix, negative quality control products, and positive quality control products; wherein the MB-qPCR Mix includes 20mM~80M Tris-HCl with a pH of 7.5~8.9, 10mM~50mM U-containing dNTP, 100mM~4M KCl, 1mM~50mM MgCl2, 0.01%~0.3% (V / V) Tween-20, 0.01%~0.1% (V / V) tetrahydrothiophene 1-oxide, 10mM~5M tetramethylammonium chloride, 10mM~100mM betaine, 0.01%~0.1% (V / V) DMSO, 0.01%~0.3% (V / V) Triton X-100, 50mM~500mM upstream primer, 50mM~500mM downstream primer, 25mM~250mM MB fluorescent probe.

[0007] Preferably, the upstream primer is a sequence as shown in SEQ ID NO.1, the downstream primer is a sequence as shown in SEQ ID NO.2, and the MB fluorescent probe is a sequence as shown in SEQ ID NO.3.

[0008] Preferably, the 5' end of the MB fluorescent probe is connected to a fluorescent luminescent group, and the 3' end is connected to a fluorescent quenching group. The fluorescent luminescent group includes one of FAM, VIC, HEX, JOE, ROX, Texas-Red, and Cy5; the fluorescent quenching group includes one of DABCYL, DABSYL, TAMRA, BHQ-1, BHQ-2, and BHQ-3.

[0009] Preferably, the Enzyme Mix includes 0.5U~2U of hot-start Taq enzyme, 0.1U~10U of UDG enzyme, 1U~10U of RNase inhibitor, 1%~50% (V / V) glycerol, 10mM~200mM potassium ion and 0.01mM~1mM EDTA.

[0010] Preferably, the positive quality control product comprises a plasmid solution synthesized using the shrimp hepatocystis target gene sequence as shown in SEQ ID NO.4.

[0011] On the other hand, the present invention also provides a method for detecting shrimp hepatoenteric cytidylcholine for non-diagnostic purposes, using the MB-qPCR detection kit for rapid detection of shrimp hepatoenteric cytidylcholine.

[0012] Preferably, the method comprises the following steps: S1: extracting sample nucleic acid; S2: Using the nucleic acid extracted in step S1 as a template, a PCR amplification reaction is performed, and the detection result is determined after the reaction is completed.

[0013] Preferably, in step S2, a 25 μL PCR amplification system is used, including 19 μL MB-qPCR Mix, 1 μL enzyme mixture, and 5 μL template.

[0014] Preferably, the PCR amplification reaction conditions are: 95°C for 3 min; 95°C for 10 s, 58°C for 30 s, and 40 cycles.

[0015] Preferably, the result is determined as follows: when the Ct value is <35 and a typical amplification curve appears, the result is positive; when 35≤Ct<37, the amplification curve is S-shaped, and the suspicious sample needs to be retested; if the retest results are consistent, it is judged to be positive for shrimp hepatoenterocytosis, otherwise it is negative for shrimp hepatoenterocytosis; when the Ct value is ≥37 or there is no amplification curve, the result is negative.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a kit for detecting shrimp hepatoenteric cysts by MB-qPCR technology, which provides a new idea for the technical field of detecting shrimp hepatoenteric cysts. The kit has the characteristics of simple operation, high specificity and high sensitivity.

[0017] Compared with the existing common PCR technology, the kit and the detection method of the present invention are simple to operate, and the result can be directly interpreted after the PCR is completed. The whole detection can detect the result within 30 minutes, which reduces the manpower, material resources and time costs. It can realize real-time monitoring of shrimp hepatoenterocystis, which is conducive to timely detection and response to virus outbreaks, and evaluate the risks of healthy shrimp farming, providing a theoretical basis for guiding scientific shrimp farming.

[0018] Furthermore, the present invention does not require subsequent experimental processing and analysis such as opening the cover, thus avoiding cross contamination between different amplification products and reducing the occurrence of false positives, and the test results are accurate and reliable.

[0019] In addition, the kit can be stored at room temperature for one week, at 4°C for half a year, and at -20°C for 2 years, and repeated freezing and thawing for more than 10 times will not cause any impact on its performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is a schematic diagram of MB fluorescent probe.

[0022] Figure 2 It is an accuracy detection amplification curve diagram of the MB-qPCR detection kit for rapid detection of shrimp hepatoenteric cytozoa of the present invention.

[0023] Figure 3 It is a sensitivity detection amplification curve diagram of the MB-qPCR detection kit for rapid detection of shrimp hepatoenteric cytozoa of the present invention.

[0024] Figure 4 It is a standard curve diagram of the MB-qPCR detection kit for rapid detection of shrimp hepatoenteric cysts of the present invention.

[0025] Figure 5 It is a specific detection amplification curve diagram of the MB-qPCR detection kit for rapid detection of shrimp hepatoenteric cytozoa of the present invention. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] The present invention designs a pair of upstream and downstream primers and a specific molecular beacon fluorescent probe (MB fluorescent probe) based on the conserved region of shrimp hepatocellular carcinoma. Figure 1 As shown, MB is a fluorescent probe with a hairpin structure. MB is designed based on the principle of fluorescence resonance energy transfer and base complementary pairing. The two ends are labeled with a fluorescent group and a quenching group respectively. In the presence of a specific target DNA, the spatial conformation of MB changes to produce fluorescence; in the absence of a specific target DNA, the fluorescent group and the quenching group are very close, and the fluorescence is quenched. MB is simple to operate, sensitive, and can perform real-time amplification detection on samples. Compared with ordinary linear oligonucleotide probes, such as Taqman probes, molecular beacons with stem-loop structures can detect mismatches, deletions, or insertion mutations of single bases in the target sequence. Therefore, the present invention is to prevent and control the spread and outbreak of shrimp hepatic enterocytes, and establishes a highly sensitive MB-qPCR detection method that can detect shrimp hepatic enterocytes, thereby achieving the purpose of detecting shrimp hepatic enterocytes.

[0030] Example 1 1. Primer design: The conserved region sequence of shrimp hepatocystis was searched from GeneBank as the target region (SEQ ID NO.4), and the primers and probes were designed using Primer 5.0 software. The primers were synthesized by Shanghai Shenggong (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.). The specific nucleotide sequences of the upstream and downstream primers and MB probe are as follows: Upstream primer: 5′-ATGCAATTAAAAGGTGGTG-3′ (SEQ ID NO. 1); Downstream primer: 5′-GAGCATCGCTTTCGCCTC-3′ (SEQ ID NO. 2); MB fluorescent probe: 5'-CCCGATG-AGAGTAGCGGAACGGATAG-CATCGGG-3' (SEQ ID NO. 3); The 3' end of the MB probe is labeled with a fluorescent quenching group, and the 5' end is labeled with a fluorescent reporter group. The fluorescent reporter group used to detect shrimp hepatoenterocystis is CY5, and the fluorescent quenching group is BHQ-2.

[0031] Target region sequence: (SEQ ID NO.4)。

[0032] 2. An MB-PCR detection kit for rapid detection of shrimp hepatoenterozoa, comprising MB-qPCR Mix, Enzyme Mix, nuclease-free pure water, positive quality control products, and negative quality control products.

[0033] The MB-qPCR Mix includes an upstream primer (SEQ ID NO.1), an oligonucleotide downstream primer (SEQ ID NO.2), an MB fluorescent probe (SEQ ID NO.3), and a PCR Probe Buffer.

[0034] The PCR Probe Buffer includes 20M Tris-HCl with a pH of 8.0, 30mM U-containing dNTP, 1M KCl, 25mM MgCl2, 0.1% (V / V) Tween-20, 0.05% (V / V) tetrahydrothiophene 1-oxide, 2M tetramethylammonium chloride, 50mM betaine, 0.06% (V / V) DMSO, and 0.1% (V / V) Triton X-100.

[0035] The Enzyme Mix includes 1U of hot-start Taq enzyme, 4U of UDG enzyme, 5U of RNase inhibitor, 30% (V / V) glycerol, 100mM potassium ion and 0.3mM EDTA.

[0036] The positive quality control product comprises a plasmid solution synthesized using the shrimp hepatoenteric cytozoon target gene sequence. Specifically, the shrimp hepatoenteric cytozoon target gene sequence is inserted into the plasmid pUC57 vector to obtain a recombinant plasmid, which is transformed into Escherichia coli DH5α for proliferation, extracted and purified, and the concentration and purity are measured by a spectrophotometer, and then quantitatively diluted with sterile TE buffer to obtain a concentration of 5.0×10 6 copies / mL of positive control.

[0037] The negative control product is Nuclease-Free Water.

[0038] Example 2 A method for detecting shrimp hepatoenteric cysts, using the kit of Example 1, specifically comprising the following steps: 1. Test samples: shrimp tissue samples 2. Nucleic acid extraction: Use a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on the silica membrane centrifugal column method or a nucleic acid extraction reagent based on the magnetic bead method, and follow the instructions of the kit to collect 80 μL of nucleic acid solution for direct testing or storage at -80°C.

[0039] 3. System establishment According to the total number of reactions N required for the test, add 19μL MB-qPCRMix, 1μL Enzyme Mix, and 5μL template to each PCR tube. Calculate the required total amount, mix well, and then dispense into special PCR reaction tubes.

[0040] Establishment and optimization of MB-qPCR test amplification conditions: (1) The primers and probes with different concentrations as shown in Table 1 were set to perform a screening experiment of primers and probes. The basic principles of the screening were: effective detection of shrimp hepatocellular carcinoma, low cost, short time consumption, etc.

[0041] After optimization, the best primer and probe concentrations were determined to be 200nM:150nM, that is, the volume ratios of the upstream primer, downstream primer, and probe were 200nM, 200nM, and 150nM, respectively.

[0042] Table 1 Primer and probe concentration optimization screening table

[0043] (2) Annealing temperature screening The PCR reaction conditions were maintained as follows: Taq enzyme activation at 95°C for 3 min, denaturation at 95°C for 10 s, and 40 cycles. Annealing extension and fluorescence acquisition were set to the 10 different Tm values ​​shown in Table 2 to perform the screening experiment for the optimal annealing temperature.

[0044] Table 2 Annealing temperature screening table

[0045] As shown in Table 2, the optimal annealing temperature Tm value after screening is 58°C, that is, the optimal PCR reaction conditions are shown in Table 3 below: Table 3 PCR amplification program

[0046] 4. Add sample Add negative quality control (Nuclease-Free Water), sample DNA solution, and positive quality control to the PCR reaction tubes containing the above reagents, with the sample volume of 5 μL. Close the tube cap tightly, mix well, and centrifuge for 3-5 seconds to collect the solution and place it at the bottom of the tube; then amplify and detect on the machine.

[0047] 5. The results are shown in Table 4 below: Table 4 Result judgment table

[0048] Test Example 1 1. Accuracy test The kit of Example 1 was used to detect nucleic acid samples of shrimp tissues that were known to be positive and negative for hepatocellular carcinoma of the shrimp according to the detection method of Example 2 to verify the accuracy of the kit.

[0049] Test results such as Figure 2 As shown, the PCR system is capable of detecting the corresponding target. After PCR amplification, the accuracy of the test result can be judged by reporting the fluorescent signal and Ct value. The positive detection rate is 100%, the negative coincidence rate is 100%, and there is no false positive or missed detection.

[0050] 2. Sensitivity test After the shrimp hepatocystis plasmid was diluted with TE buffer, the value was determined using Nanodrop and Qubit, and the result was 1×10 12 copies / μL, and then converted to copies / mL and performed gradient dilution. Eight concentration gradients were set, namely E5: 1×10 5 copies / mL, E4: 1×10 4 copies / mL, E3: 1×10 3 copies / mL, E2: 1×10 2 10 copies / mL, E1: 10 copies / mL, 0.5E1: 5 copies / mL, 0.25E1: 2.5 copies / mL, E0: 1 copies / mL and NTC (negative control H2O), 10 replicate samples were run for each concentration to verify the sensitivity of the detection method described in Examples 1 and 2 of the present invention.

[0051] Test results such as Figure 3 As shown in the figure, the shrimp hepatocystis can be accurately detected at a sample concentration of 2.5 copies / mL, indicating that the detection method has a high sensitivity and the detection limit is 2.5 copies / mL. At the same time, a standard curve was drawn. Figure 4 shown.

[0052] Specificity Detection DNA of other non-target shrimp parasites (nematodes, microsporidia, anchor worms, and cluster worms) was used as specific detection samples and detected using the detection methods described in Examples 1 and 2. The detection results are as follows: Figure 5 As shown, the results showed that no specific amplification occurred, indicating that the kit has strong specificity. The amplification curve in the figure is a positive control.

[0053] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma, characterized in that: The kit includes MB-qPCR Mix, Enzyme Mix, negative quality control products, and positive quality control products; wherein the MB-qPCR Mix includes 20mM~80M Tris-HCl with a pH of 7.5~8.9, 10mM~50mM U-containing dNTP, 100mM~4M KCl, 1mM~50mM MgCl2, 0.01%~0.3% (V / V) Tween-20, 0.01%~0.1% (V / V) tetrahydrothiophene 1-oxide, 10mM~5M tetramethylammonium chloride, 10mM~100mM betaine, 0.01%~0.1% (V / V) DMSO, 0.01%~0.3% (V / V) Triton X-100, 50mM~500mM upstream primer, 50mM~500mM downstream primer, 25mM~250mM MB fluorescent probe.

2. The MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma according to claim 1, characterized in that: The upstream primer is a sequence shown in SEQ ID NO.1, the downstream primer is a sequence shown in SEQ ID NO.2, and the MB fluorescent probe is a sequence shown in SEQ ID NO.

3.

3. The MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma according to claim 2, characterized in that: The 5' end of the MB fluorescent probe is connected to a fluorescent group, and the 3' end is connected to a fluorescent quenching group; the fluorescent group includes one of FAM, VIC, HEX, JOE, ROX, Texas-Red, and Cy5; the fluorescent quenching group includes one of DABCYL, DABSYL, TAMRA, BHQ-1, BHQ-2, and BHQ-3.

4. The MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma according to claim 1, characterized in that: The Enzyme Mix includes 0.5U-2U of hot start Taq enzyme, 0.1U-10U of UDG enzyme, 1U-10U of RNase inhibitor, 1%-50% (V / V) glycerol, 10mM-200mM potassium ion and 0.01mM-1mM EDTA.

5. The MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma according to claim 1, characterized in that: The positive quality control product includes a plasmid solution synthesized using the shrimp hepatocystis target gene sequence shown in SEQ ID NO.

4.

6. A method for detecting hepatocellular carcinoma of shrimp for non-diagnostic purposes, characterized in that: The MB-qPCR detection kit for rapid detection of shrimp hepatocellular carcinoma according to any one of claims 1 to 5 is used.

7. The method according to claim 6, characterized in that The method comprises the following steps: S1: extracting sample nucleic acid; S2: Using the nucleic acid extracted in step S1 as a template, a PCR amplification reaction is performed, and the detection result is determined after the reaction is completed.

8. The method according to claim 7, characterized in that In step S2, a 25 μL PCR amplification system is used, including 19 μL MB-qPCR Mix, 1 μL enzyme mixture, and 5 μL template.

9. The method according to claim 7, characterized in that: The PCR amplification reaction conditions are: 95°C for 3 min; 95°C for 10 s, 58°C for 30 s, and 40 cycles.

10. The method according to claim 7, characterized in that The result is determined as follows: when the Ct value is <35 and a typical amplification curve appears, the result is positive; when 35≤Ct<37, the amplification curve is S-shaped, and the suspicious sample needs to be retested; if the retest results are consistent, it is judged to be positive for shrimp hepatocystis, otherwise it is negative for shrimp hepatocystis; when the Ct value is ≥37 or there is no amplification curve, the result is negative.

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